沉默p53和p21基因延缓髓核细胞衰老退变实验研究

目的髓核细胞衰老凋亡是椎间盘退行性变的病理基础,探讨髓核细胞表型分子及延缓髓核细胞衰老退变的机制。方法原代培养8～10周龄雄性SD大鼠髓核细胞,免疫细胞化学染色鉴定髓核细胞表型分子低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)、HIF-1β、基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)及Ⅱ型胶原表达。小干扰RNA(small interference RNA,siRNA)瞬时转染髓核细胞沉默p53、p21后行RT-PCR及Western blot检测沉默效率,siRNA转染前后细胞衰老相关β半乳糖苷酶(senescence associated-β-galactosidase,SA-β-gal)染色检测髓核细胞衰老变化,流式细胞仪检测髓核细胞周期变化,MTT法生长曲线分析髓核细胞增殖变化。结果免疫细胞化学染色显示髓核细胞表达HIF-1α、HIF-1β、MMP-2及Ⅱ型胶原。第35代髓核细胞转染p53、p21 siRNA后RT-PCR及Western blot检测示p53、p21表达明显受到抑制。第35代髓核细胞SA-β-gal染色阳性率明显高于第1代髓核细胞(P<0.001);正常第35代髓核细胞经p53 siRNA(p53转染组)和p21 siRNA(p21转染组)转染后,SA-β-gal阳性率明显低于正常第35代髓核细胞(正常组)和加入脂质体LipofectaminTM2000而无siRNA的第35代髓核细胞(阴性对照组),差异有统计学意义(P<0.001)。细胞周期分析显示,p53转染组及p21转染组G1期百分比均明显低于正常组和阴性对照组(P<0.05),S期百分比明显高于正常组和阴性对照组(P<0.05)。MTT生长曲线显示转染p53、p21 siRNA后可促进髓核细胞增殖。结论沉默p53、p21基因可通过调节细胞周期而抑制髓核细胞衰老退变,改善椎间盘退变过程;沉默p53、p21基因可能是潜在的治疗椎间盘退行性变的方法。

Effect of silencing p53 and p21 on delaying senescence of nucleus pulposus cells

Objective The senescence and death of nucleus pulposus(NP) cells are the pathologic basis of intervertebral disc degeneration(IVD).To investigate the molecular phenotypes and senescent mechanism of NP cells,and to identify the method of alleviating senescence of NP cells.Methods The primary NP cells were harvested from male Sprague Dawley rats(8-10 weeks old);the hypoxia inducible factor 1α(HIF-1α),HIF-1β,matrix metalloproteinase 2(MMP-2),and collagen type II as phenotypic markers were identi ed through immunocytochemical staining.RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA(siRNA).Senescence associated-β-galactosidase(SA-β-gal) staining was used to test the senescence of NP cells,ow cytometry to test the change of cell cycle,the growth curve analysis to test the NP cells proliferation.Results Immunocytochemical staining showed that NP cells expressed HIF-1α,HIF-1β,MMP-2,and collagen type II.RT-PCR and Western blot showed that the relativeffexpressions of mRNA and protein of p53 and p21 were signi cantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA.The percentage of SA-β-gal-positive NP cells at passage 35 was signi cantly higher than that at passage 1(P < 0.001).And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were signi cantly lower than that in control group(P < 0.001).The ow cytometry showed that the G1phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was signi cantly shorter than that in control group(P < 0.05),but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were signi cantly longer than that in control group(P < 0.05).In addition,the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA.Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21.The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.